According to the WHO definition, atherosclerosis is a combination of changes in the intimae of the arteries resulting from focal accumulation of lipids and complex compounds, accompanied by fibrous tissue formation, calcification and in turn associated with changes in the media.
Atherosclerosis may be considered as a special form of arteriosclerosis with pathogenic significant deposition of lipids in the arterial wall. Most forms of arteriosclerosis involve fatty degeneration of vascular wall, the terms “arteriosclerosis” and “atherosclerosis” may be used synonymously (Assmann G. in “Lipid Metabolism and Atherosclerosis” Schattauer Verlag GMbH, Stuttgart 1982: 1).
Lipids are insoluble in aqueous solutions. Lipoproteins are the particles enabling transport of the lipids in the blood. Lipoproteins are divided into various categories according to their density, depending on how they can be separated by ultracentrifugation (Havel R J et al. J Clin Invest 1955, 34:1345). Low density-lipoproteins (LDL) (d=1.019-1.063 g/mL) transport the bulk of the cholesterol in the blood. They are composed of about 75% lipid (primarily cholesterol, cholesteryl esters and phospholipids), approximately 70% of the total cholesterol in the blood is transported by LDL particles.
Hypercholesterolemia is used to reflect a rise in plasma cholesterol higher than level considered normal for a particular population and is one of the critical factors in the onset and the progress of atherosclerosis. More than half of all deaths in Western society are related to atherosclerosis cardiovascular diseases (Murray C J L and Lopez A D. Lancet 1997; 349:1269-1276).
Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease produced in the receptor gene of the LDL (LDL-r) this gene codifies a protein that allows the intracellular uptake and degradation of the LDL (Goldstein J L, Brown M S Ann Rev Cell Biol 1985; 1:1-39).
The penetrance of FH is almost 100% meaning that half of the offspring of an affected parent has a severely elevated plasma cholesterol level from birth onwards, with males and females equally affected (Goldstein J L, Brown M S. The metabolic basis of inherited disease. Scriver C R, Beaudet A L, Sly W S, Valle D, eds. McGraw Hill New York 6th edition, 1989; 1215-1250).
FH affected individuals display arcus lipoides corneae, tendon xanthomas and premature symptomatic coronary heart disease (Scientific Steering Committee on behalf of the Simon Broome register Group. Atherosclerosis 1999; 142: 105-115). FH is one of the most common inherited disorders with frequencies of heterozygote patients of and homozygote estimated to be 1/500 and 1/1,000,000, respectively.
Certain populations, such as a small number of mutations predominate due to founder effects and therefore, the frequency of heterozygous FH is higher, these populations include French Canadians (Leitersdorf E et al. J Clin Invest 1990; 85:1014-1023), Christian Lebanese (Lehrman M A et al. J Biol Chem 1987; 262:401-410) Druze (Landsberger D et al. Am J Hum Genet 1992; 50: 427-433) Finns (Koivisto U M et al. J Clin Invest 1992; 90:219-228) South African Afrikaner (Kotze M J et al. Ann Hum Genet 1991; 55:115-121), and Ashkenazi Jews of Lithuanian descent (Meiner V et al. Am J Hum Genet 1991; 49:443-449) have the peculiarity that they have only a few mutations responsible for the FH, result of founder effects and therefore the frequency of heterozygous FH in those populations is higher than the estimate for other populations.
FH heterozygous patients display a very high plasma cholesterol concentration, generally above the 95th percentile value. In patients with FH the age-standardised and sex-standardised mortality ratios are four to five times higher than in the general population (Scientific Steering Committee on behalf of the Simon Broome Register Group. Atherosclerosis 1999; 142: 105-115). Patients who have inherited two mutant at the LDL-r locus are termed “FH homozygotes” or “FH compound heterozygotes”, in which case those are practically no functional receptors which lead to a six-fold to eight-fold elevation in plasma LDL-c levels above normal. In the majority of these patients, a coronary heart disease typically occurs before the age of 20 years (Goldstein J L et al. N Engl J Med 1983; 309:288-296). If individuals with heterozygous or homozygous FH could be diagnosed before they develop symptomatic disease, they could be treated preventively to substantially reduce their risk of myocardial infarction.
The LDL-r is an ubiquitous trans-membrane glycoprotein of 839 amino acids that mediates the transport of LDL into cells via endocytosis (Goldstein J and Brown M J Biol Chem 1974; 249:5153-5162) (FIG. 1).
The LDL-r gene lies on the short arm of chromosome 19p13.1-13.3 (Yamamoto T et al. Cell 1984; 39: 27-38), spans 45,000 base pairs (bp). It comprises 18 exons and 17 introns encoding the six functional domains of the mature protein: Signal peptide, ligand-binding domain, epidermal growth factor (EGF) precursor like, O-linked sugar, trans-membrane and cytoplasmic domain (Sundhof T et al. Science 1985; 228:893-895) (FIG. 2).
The LDL-r synthesis is regulated by a sophisticated feedback mechanism that controls the transcription of the LDL-r gene in response to variations in the intracellular sterol concentration and the cellular demand for cholesterol (Sudhof T C et al J Biol Chem 1987; 262:10773-10779). DNA motifs necessary for transcriptional regulation of the LDL-r gene are located within 177 bp of the proximal promoter (Sudhof T C et al. J Biol Chem 1987; 262: 10773-10779). This region contains all the cis-acting elements for basal expression and sterol regulation and includes three imperfect direct repeats of 16 bp each. Repeat 1 and 3 containing binding sites for the transcriptional factor Sp1 and are essential for producing the basal expression of the gene but require the contribution of the repeat 2 for full expression (Dawson P A et al. J Biol Chem 1988; 263; 3372-3379). Repeat 2 contains a 10 bp regulatory element, SRE-1, (Smith J R et al. J Biol Chem 1990; 265:2306-2310) that allows binding of the transcriptional factor designated as SREBP-1, when the intra-cellular sterol concentration diminishes. To date, several naturally-occurring mutations have been mapped to the transcriptional regulatory elements of the LDL gene receptor (Hobbs H H, et al. Hum Mutat 1992; 1:445-466; Koivisto U M, et al ProcNatl Acad Sci USA, 1994; 91:10526-10530), Mozas P, et al J Lipid Res 2002; 43:13-18, (worldwideweb: ucl.ac.uk/fh; worldwideweb:umd.necker.fr.)
Exon 1 encodes the signal peptide, a sequence of 21 amino acids, which is cleaved from the protein during the translocation into the endoplasmic reticulum. Several frameshift, missense and nonsense mutation has been described in this exon (worldwideweb: ucl.ac.uk/fh; worldwideweb: umd.necker.fr.)
Exons 2 to 6 encode the ligand binding domain, which consists of seven tandem repeats of 40 amino acids each. The structure of the ligand binding domain has been partially elucidated (Jeon H et al. Nature Struc Biol 2001; 8:499-5049). There are a cluster of negatively charged amino acids, Asp-X-Ser-Asp-Glu in each repeat and six cysteine residues that form three disulfide bonds.
The second domain of the human LDL-r consists of 400 amino acid sequence, encoded by exons 7 to 14. This sequence shows a 33% of homology of the epidermal growth factor precursor (EGFP). Like the ligand binding domain, this region also contains three repeats of 40 amino acids with cysteine-rich sequences. The first two repeats, designated A and B, are contiguous and separated from the third repeat, by a 280 amino acid region that contains five copies of the YWTD (Tyr-Trp-Thr-Asp) sequence. The EGFP like domain is fundamental for the acid-dependent dissociation of the LDL particles from the LDL-r and clathrin coat pits that takes place in the endosome during receptor recycling. Of the all mutations described to date, approximately 55% are located in the EGFP-homology region and 35% among the YWTD repeats worldwideweb: ucl.ac.uk/fk worldwideweb:umd.necker.fr.)
The third domain of the LDL-r that is encoded by exon 15, is a region rich in threonine and serine residues. The function of this domain is unknown, but it is known that in this region the carbohydrate chains are anchored. This region show minimal sequence conservation among six species analysed and it is thought that this domain play a role in the stabilization of the receptor (Goldstein et al. In The Metabolic and Molecular Basis of Inherited Disease. Sciver C R, Beaudet A L, Sly W S, Valle D. 7th Edition. McGraw Hill, 1995: 1981-2030).
The trans-membrane domain comprises 22 hydrophobic amino acids coded by exon 16 and the 5′ end of exon 17. This domain is essential for anchoring the LDL-r to the cell membrane.
The cytoplasmic domain of the LDL-r, is formed by a sequence of 50 amino acids residues, is encoded by the 3′ region of the exon 17 and the 5′ end of the exon 18. This domain contains two sequence signals for targeting the protein to the cell surface and for localizing the receptor in coated pits (Yokode M, et al. J Cell Biol 1992; 117: 39-46). This domain is one of the most conservedm with a percentage of amino acids converved of 86% among six species analysed.
LDL-r mutations found in FH patients, have been classified into 5 classes: null alleles, transport defective alleles, binding defective alleles, internalization-defective alleles and recycling-defective alleles. As a general rule, each category is associated with mutations localised in a region of the gene that codes for one particular domain of the protein (Hobbs, H H, et al. Hum Mutat 1992; 1:445-466).
The heterogeneity in FH patients in relation to plasma LDL-c levels and coronary heart disease is due in part to differences in the nature of the mutation (Sun X M et al. Arterioscler Thromb Vas Biol 1993; 13:1680-1688, Kotze M J et al. Arterioscler Thromb Vas Biol 1993; 13:1460-1468; Gudnason V et al. Arterioscler Thromb Vas Biol 1997; 17:3092-3101). On the other hand, in FH heterozygote patients, the LDL-c lowering response after treatment with hydroxy-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors depends in part on the nature of the mutation in the LDL-r gene (Leisterdorf E et al. Circulation 1993; 87:35-44; Jeenah M et al. Atherosclerosis 1993; 98:51-58, Sijbrands E J G et al. Atherosclerosis 1998; 136: 247-254).
The primary ligand for the receptor is LDL, which contains a single copy of a protein called apolipoprotein B-100 (ApoB-100) (Goldstein J and Brown M J Biol Chem 1974; 249:5153-5162). This apolipoprotein has a zone rich in basic amino acids and being the site where binding to the receptor (Borén J et al. J Clin Inves 1998; 101: 1084-1093). Several mutations located in the apolipoprotein B gene have been found altering the functional activity of the protein and decreasing its capacity for withdrawal LDL particles, and leading to accumulation of LDL cholesterol in plasma. To date, four mutations have been identified in the apo B-100 gene which cause a hypercholesterolemia named Familial Defective (BDF) apolipoprotein, all of them located in the LDL-r binding domain of the apo B-100 protein (residues 3130-3630): R3480W, R3500Q, R3500W and R3531C (Soria L et al. Proc Natl Acad Sci USA 1989; 86: 587-591; Pullinger C R, et al. J Clin Invest 1995; 95:1225-1234; Gaffney D, et al. Arterioscler Thromb Vasc Biol 1995; 15:1025-1029; Boren J, et al. J Biol Chem 2001; 276; 9214-9218). The CGG-to-CAG mutation at codon for amino acid 3500, resulting in a glutamine substitution for arginine (R3500Q), is the most frequent alteration causing Familial Defective apolipoprotein B-100 (FDB). Patients heterozygous for the apoB-3500 mutation are usually hypercholesterolemic, although serum cholesterol concentrations can vary from those found in FH to only modest elevations (Tybjaerg-Hansen A, et al. Atherosclerosis 1990; 80:235-242; Hansen P S, et al. Arterioscl Throm Vasc Biol 1997; 17:741-747). Since clinical and biochemical characteristics in those patients are very similar, the differential diagnostic between patients with FDB or FH is only possible by genetic molecular diagnosis.
The clinical diagnosis of FH is based on the analytical data of lipids and lipoproteins in the plasma, clinical symtomatology (xanthomas) and family and personal coronary disease history. The WHO, through its MedPed program recommends a series of criteria be followed to perform the clinical diagnosis of FH. These criteria are based on a scoring system relying on the personal and family history of hypercholesterolemia, of the patient's clinical and analytic characteristics. When the punctuation reached by the patient is equal to or higher than 8 points the clinical criterion of FH diagnosis is classed as “certain”, between 5 and 8 points as “likely and between 3 and 5 points as “possible” (Familial Hypercholesterolemia. Report of a second WHO consultation. The International MedPed FH Organization, Geneva 1998). However, some patients do not fulfil the FH criteria, because the family history is incomplete or unknown, or because at the time of the analysis they presented only moderate concentrations of plasmatic cholesterol and lacked signs of tissue cholesterol deposition, as tendinosous xanthomas, arcus corneae or xanthelasmas.
In families whose mutation of the r-LDL gene is known, it has been demonstrated that the best “cut-off” point for the diagnosis is use of the 90th percentile for the c-LDL concentration (Umans-Eckenhausen MAW et al. Lancet 2001; 357:165-168. However, 18% of FH patients carriers of the mutation have a total cholesterol concentration below this percentile and moreover the proportion of false positives was 18%. Therefore, there will be a high percentage of wrong diagnoses if only the plasmatic cholesterol figure is utilized. It has been published that more than 50% of patients do not receive lipid lowering therapy and dietary counseling as a result of not having been diagnosed correctly as patients with FH (Williams R R et al., Am J Cardiol. 1993; 72:18 D-24D).
The elucidation of the molecular basis of FH has made diagnosis at the DNA level feasible in the vast majority of cases. Demonstration of an underlying defect in the LDL-r gene, constitutes in fact the definite confirmation of the diagnosis (Familial hypercholesterolemia. Report of a second WHO consultation. The International MedPed FH Organization, Geneva 1998). Although accurate diagnosis of FH is possible by means of molecular methods, their use in heterogeneous populations is limited at present owing to mutational heterogeneity of the LDL-r gene.
In application PCT WO-88/03175 (Biotechnology Research Partners, Ltd.) a method is claimed for the diagnosis of atherosclerosis, based on detection of the presence or absence of various polymorphisms in the gene region of the apolipoprotein AO-CIII-AIV, or in the genes apoB, apoCI, apoAII, as well as in the LDL receptor gene. Specifically for this gene, utilization of the polymorphisms Cfr131 and BstEII is presented.
Another document of interest is Japanese patent JP-10099099 which refers to the use of a mutation in the codifier triplet of the amino acid 109, specifically the insertion of a C, for the diagnosis of abnormalities in the LDL receptor gene, although familial hypercholesterolemia is not specifically mentioned.
Finally, U.S. Pat. No. 4,745,060 and U.S. Pat. No. 4,966,837, both of the University of Texas, present methods for the diagnosis of familial hypercolesterolemia on the basis of mutations in the LDL receptor gene. However, what is claimed in the first of them are sequences corresponding to the “normal” gene, presenting a particular example of a mutation that is defined by the restriction map change with Xba I. In the second patent, on its part, the use of various restriction enzymes is claimed (Eco RI, Asp 718, Taq I, Bam HI, Xba I, Inf. I, Bgl II, Cla I, Eco RV, Kpn I, Pvu II, Sph I, Sst I, Sst II, Stu I, Xho I, Nde I and Nsi I) in a method for determining mutations in the LDL-r gene, based on observing the alteration of the restriction model with these enzymes compared to the model corresponding to the normal gene.
The closest patent document to the invention is WO02/06467, in which a method is described, for the detection of errors in the lipidic metabolism based on a series of mutations and polymorphisms of the LDL-r gene. However, none of the mutations or polymorphisms described in said patent coincides with those claimed in the present application.